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Rapid Purification of a New Humanized Single-chain Fv Antibody/Human Interleukin-2 fusion protein Reactive against HER2 Receptor

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 707-712页

作者： Tak-Chun YIP Cheuk-Sang KWOK Department of Clinical Oncology Queen Elizabeth Hospital Hong Kong China Division of Radiation Oncology City of Hope National Medical Center Duarte CA91010 USA

Human embryonic kidney 293 cells were transfected with plasmid pcDNA-H520C9scFv-rhIL- 2 containing a chimeric cDNA encoding the humanized 520C9 scFv/recombinant human IL-2 fusion protein (H520C9scFv-rhIL-2). The transfected cells in plateau growing phase were cultured in serum-free medium for three days. The supernatant was collected, concentrated and purified using an affinity column packed with CNBr-activated Sepharose 4B coupled with anti-rhIL-2 mouse monoclonal antibody. The purified fusion protein was analyzed by ELISA, SDS-PAGE and Western blot. The fusion protein showed only one band in both silver stained electrophoresis gel and Western blot developed by ECL chemiluminescence system. Its molecular weight was confirmed to be about 45 kD. This fusion protein possessed binding specificity against p185 positive SKOV3 and B16/neu cells, and it might stimulate IL-2-dependent CTLL-2 cell prolifera- tion as well.

关键词： single-chain Fv antibody IL-2 fusion protein human embryonic kidney 293 cells affinity chromatography

VEGF-B antibody and interleukin-22 fusion protein ameliorates diabetic nephropathy through inhibiting lipid accumulation and inflammatory responses

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Acta Pharmaceutica Sinica B 2021年第1期11卷 127-142页

作者： Yilan Shen Wei Chen Lei Han Qi Bian Jiajun Fan Zhonglian Cao Xin Jin Tao Ding Zongshu Xian Zhiyong Guo Wei Zhang Dianwen Ju Xiaobin Mei Department of Nephrology Changhai HospitalSecond Military Medical UniversityShanghai 200433China Department of Biological Medicines Fudan University School of PharmacyShanghai 201203China Department of Nephrology Shanghai Yangpu Hospital of TCMShanghai 200090China Department of Ophthalmology Stanford University School of MedicinePalo AltoCA 94304USA

Diabetic nephropathy(DN)is considered the primary causes of end-stage renal disease(ESRD)and is related to abnormal glycolipid metabolism,hemodynamic abnormalities,oxidative stress and chronic inflammation.Antagonism of vascular endothelial growth factor B(VEGF-B)could effi-ciently ameliorate DN by reducing renal lipotoxicity.However,this pharmacological strategy is far from satisfactory,as it ignores numerous pathogenic factors,including anomalous reactive oxygen species(ROS)generation and inflammatory responses.We found that the upregulation of VEGF-B and downregulation of interleukin-22(IL-22)among DN patients were significantly associated with the progression of DN.Thus,we hypothesized that a combination of a VEGF-B antibody and IL-22 could protect against DN not only by regulating glycolipid metabolism but also by reducing the accumulation of inflammation and ROS.To meet these challenges,a novel anti-VEGFB/IL22 fusion protein was developed,and its therapeutic effects on DN were further studied.We found that the anti-VEGFB/IL22 fusion protein reduced renal lipid accumulation by inhibiting the expression of fatty acid transport proteins and ameliorated inflammatory responses via the inhibition of renal oxidative stress and mitochondrial dysfunction.Moreover,the fusion protein could also improve diabetic kidney disease by increasing insulin sensitivity.Collectively,our findings indicate that the bifunctional VEGF-B antibody and IL-22 fusion protein could improve the progression of DN,which highlighted a novel therapeutic approach to DN.

关键词： Diabetic nephropathy Vascular endothelial growth factor B Interleukin-22 fusion protein

Transgenic plants expressing the AalT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests

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Insect Science 2016年第2期23卷 265-276页

作者： Shu-Min Liu Jie Li Jin-Qi Zhu Xiao-Wei Wang Cheng-Shu Wang Shu-Sheng Liu Xue-Xin Chen Sheng Li Key Laboratory of Insect Developmental and Evolutionary Biology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai State Key Laboratory of Rice Biology and Ministry of Agriculture Key Laboratory of Agricultural Entomology Institute of Insect Sciences Zhejiang University Hangzhou China

The adoption of pest-resistant transgenic plants to reduce yield losses and de- crease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AalT/GNA, in which an insecticidal scor- pion venom neurotoxin （Androctonus australis toxin, AalT） is fused to snowdrop lectin （Galanthus nivalis agglutinin, GNA）. Compared with transgenic tobacco and Arabidop- sis plants expressing AaIT or GNA, transgenic plants expressing AalT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AalT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AalT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AalT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

关键词： AalT AalT/GNA chewing pest GNA fusion protein pest control resistance sucking pest toxicity transgenic plant

Efficient growth inhibition of ErbB2-overexpressing tumor cells by antiErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo

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Acta Pharmacologica Sinica 2007年第10期28卷 1611-1620页

作者： Ming SHI~1,Ling ZHANG~2,Hong-tao GU~2,Feng-qin JIANG~2,Lu QIAN~1,Ming YU~1,Guo-jiang CHEN~1,Qun LUO~3,Bei-fenSHEN~1,Ning GUO~(1,4)~1 Institute of Basic Medical Sciences,Beijing 100850,China ~2 Institute of Basic Medical Sciences,The Academy of Medical Science of ShandongProvince,Ji-nan 250062,China ~3 301 Hospital,Beijing 100853,China Institute of Basic Medical Sciences Beijing China Institute of Basic Medical Sciences The Academy of Medical Science of Shandong Province Ji-nan China 301 Hospital Beijing China

Aim:To investigate the antitumor activities of an anti-ErbB2 scFv-Fc-interleukin2 (IL-2) fusion protein (HFI) in vitro and in vivo.Methods:fusion protein HFI wasconstructed.The efficacy of HFI in mediating tumor cell lysis was determined bycolorimetric lactate dehydrogenase release assays.The antitumor activity of HFIwas evaluated in tumor xenograft models.Results:The fusion protein was foldedas a homodimer formed by covalently linking Fc portions and it retained ErbB2specificity and IL-2 biological activity.HFI mediated antibody-dependent cell-mediated cytotoxicity (ADCC) at low effector-to-target ratios in vitro and im-proved the therapeutic efficacy of IL-2 in experiments in vivo.Conclusion:Thegenetically-engineered anti-ErbB2 scFv-Fc-IL-2 fusion protein exhibited high ef-ficiency both in mediating ADCC in vitro and significant antitumor activity intumor xenograft models.

关键词： ErbB2 antibody interleukin-2 fusion protein tumor

Diphtheria Toxin/Human B-Cell Activating Factor fusion protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

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Chinese Journal of Cancer Research 2012年第3期24卷 238-244页

作者： Xin-pu Gao Zheng-min Liu Yu-lian Jiao Bin Cui Yue-ting Zhu Jie Zhang Lai-cheng Wang Yue-ran Zhao Central Laboratory Provincial Hospital Affiliated to Shandong UniversityJinan 250021China Laboratory Department of Jinan Blood Station Blood Center of Shandong ProvinceJinan 250001China

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

关键词： B cell-activating factor B-lineage acute lymphoblastic leukemia Diphtheria toxin fusion protein

Expression, Purification, and Refolding of Recombinant fusion protein hIL-2/mGM-CSF

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Biomedical and Environmental Sciences 2008年第6期21卷 509-513页

作者： QIAN WEN LI MA WEI LUO MING-QIAN ZHOU XIAO-NING WANG Institute of Molecular Immunology Southern Medical University Guangzhou 510515 Guangdong China School of Biosciences and Bioengineering South China University of Technology Guangzhou 510641 Guangdong China

Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF）（hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.

关键词： HIL-2/mGM-CSF fusion protein Purification Refolding

Binding of Divalent H-2K^(d)/IgG2aFc fusion protein to Murine Macrophage via Fc-FcR Interaction

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Cellular & Molecular Immunology 2007年第2期4卷 147-151页

作者： Wei Xiao Xueling Chen Lin Zhou Shengjun Lu Zhihui Liang Xiongwen Wu Department of Immunology Tongji Medical College Huazhong University of Science ＆ Technology Wuhan 430030 China Department of Immunology School of Medicine Shihezi University Shihezi 832002 China

Peptide-MHC class I complex （pMHC） is a specific ligand for TCR recognition, and important for CD8^＋T cell activation. Here we described a genetically engineered divalent class I major histocompatibility complex （MHC） molecule, H-2K^d/IgG2aFc, a fusion protein consisting of the extracellular domains of H-2K^d, a murine MHC class I molecule, and the Fc region of IgG2a. This fusion protein is expected to attach the H-2K^d molecule to the surface of murine macrophage （MФ） through its Fc portion binding to Fc receptor （FcR） of MФ. cDNAs coding for the extracellular domains of H-2K^d and the Fc region of IgG2a were cloned respectively, and then recombined into plasmid pcDNA3.1（＋）. The H-2K^d/IgG2aFc protein was expressed by the plasmid-transfected cell line J558L, and purified from its supernatant with a Staphylococcal protein A （SPA） column. The fusion protein showed a 58.4 kDa band as revealed by SDS-PAGE and Western blotting with murine IgG-specific antibody, which consists with that expected for extracellular domains of H-2K^d heavy chain plus the Fc region of IgG2a. The sandwich ELISA assay with antibodies specific for Fc portion and for H-2K^d indicated the fusion protein consists of both Fc portion and H-2K^d. Peritoneal MФ of C57BL/6 （H-2K^b） can be stained with H-2K^d specific monoclonal antibody （mAb） after incubated with the H-2K^d/IgG2aFc fusion protein. These results demonstrate the fusion protein can be used to attach the H-2K^d molecule to the surface of murine MФ, and provides a novel means to manipulate the T cell recognized epitope on the surface of murine MФ, which can be applied to activate antigen-specific cytotoxic T lymphocyte （CTL）.

关键词： H-2K^d/IgG2aFc divalent MHC fusion protein Fc receptor

Preparation of ChlL-2 and IBDV VP2 fusion protein by Baculovirus Expression System

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Cellular & Molecular Immunology 2005年第3期2卷 231-235页

作者： Yan Liu Yongwei Wei Xiaofeng Wu Lian Yu Department of Biomedical Engineering Zhejiang University Hangzhou310027 China Zhejiang Province Key Lab of Preventive Veterinary Medicine China College of Animal Sciences Zhejiang University Huajiachi CampusHangzhou 310029 China

This study aims to produce an effective subunit vaccine against infectious bursal disease virus （IBDV）. The genes of chicken interleukin-2 （ChIL-2） and IBDV viral protein 2 （VP2） were amplified and fused by splice overlap extension-polymerase chain reaction （SOE-PCR）. The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus （HyNPV） genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. fusion protein VP2-IL2 was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry. Cellular ＆ Molecular Immunology. 2005;2（3）：231-235.

关键词： IBDV ChIL-2 fusion gene fusion protein

Promotion of Chondrogenesis of Marrow Stromal Stem Cells by TGF-β3 fusion protein In Vitro

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2013年第5期33卷 692-699页

作者：吴薇但洋杨述华杨操邵增务许伟华李进刘先哲郑东 Department of Pediatics Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Department of Orthopedics Union Hospital Tongji Medical College Huazhong University of Science and Technology

The purpose of this study was to investigate the repair of the osteoarthritis（OA）-induced car- tilage injury by transfecting the new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted ther- apy function into the bone marrow-derived mesenchymal stem cells （MSCs） in rats. The recombinant of plRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector plRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat em- bryos by RT-PCR and inserted into the upstream and downstream of MMP from plRES-EGFP-MMP respectively, so as to construct the recombinant plasmid ofplRES-EGFP-LAP-MMP-mTGF-β3, plRES- EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cul- tured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction （ACLT）. The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Gre- en and graded by Mankin＇s scale, plRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein （39 kD） was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.

关键词： fusion protein marrow stromal cells directional differentiation transforming growth fac-tor-beta 3 cartilage injury